Cell Culture Contamination Troubleshooting

Overview
Types of Contamination
Common Causes and Prevention of Contaminants
   Microbial (bacteria, fungi, yeast)
   Mycoplasma
   Viral Contamination
   Chemical Contamination
General Tips and Techniques
   Working within the Biological Safety Cabinet
   Pipetting and Prevention of Aerosols
   Disinfection
   How to Perform Routine Monitoring of Healthy Cells

Culture Contamination: An Overview

Although "cell culture contamination" typically conjures thoughts of bacteria among cells and cloudy media, unwanted invaders in the culture flask can take many forms. Viral and chemical contaminants can also have considerable consequences for culture health, and ensuring that cell lines are not cross-contaminated is critical to reproducibility of results.

How pervasive is cell contamination? Based on studies by the FDA, ATCC, and others, it is estimated that 5 – 30% of all cell cultures today are contaminated with mycoplasma species alone. Incidence of viral contamination of common cell lines exceeded 25% in one study⁵, and non-cytopathic viruses are even more likely than mycoplasma to escape detection, as culture health may not provide clues to their presence.

Figure 1. Cell Contamination

The key to controlling a specific contaminant is sometimes related to the ease with which it is detected. Bacterial, fungal, and yeast contaminants are often visible to the unaided eye, and may quickly kill the cells in culture, but subtle morphology may make significant microbial intruders like mycoplasma more difficult to identify. By comparison, viruses cannot be detected at all by conventional light microscopy, and their discovery is therefore often prompted by observations of unexplained detachment or other signs of poor cell health or even cell death.

One of the most significant concerns confronting the biology community in recent years is the contamination or even outright misidentification of research cell lines. For more on this important topic, click here to understand the causes and prevalence of cross-contaminated cell lines, and how to evaluate the integrity of cell line identity.

Types of Culture Contamination

Bacteria, Fungi, Yeast:
Microbial Contaminants

Bacterial, fungal (including molds), and yeast contamination are usually visible to the unaided eye as rapid-onset turbidity and color change of the culture medium (provided that the medium is supplemented with phenol red, the most common non-toxic pH indicator). Standard light microscopy will also reveal bacterial cells and fungal structures, so daily microscopic observation of cultures will ensure early detection of microbial contamination and enable appropriate action to be taken as soon as the first signs become apparent. In particular, prompt removal of contaminated cultures is needed to protect neighboring cultures, as well as the sterile tissue culture environment including culture incubators, baths, and the biosafety cabinet. Additionally, specific testing for bacteria and fungi should be used as part of a routine and regular quality control screening procedure.

Common Causes and Prevention of Microbial Contamination

Root / Cause	Prevention
Technique:
Manipulations, pipetting, dispensing
Nonsterile surfaces and equipment	Clear work area of items not in immediate use.
Spillage on necks and outside of bottles and on work surface	Swab regularly with 70% alcohol. Do not pour liquids. Dispense by pipette, autodispenser, or transfer device. If pouring is unavoidable: (1) do so in one smooth movement, (2) discard the bottle that you pour from.
Touching or holding pipette too close to tip, touching necks of bottles, inside screw caps	Hold pipettes above graduations. Handle sterile pipettes by overwrap sleeve.
Splash-back from waste beaker	Discard waste into a beaker with a funnel or, use vacuum to draw off waste whenever possible.
Sedimentary dust or particles of skin settling on the culture or bottle; hands or apparatus held over an open plate, bottle, dish	Never pass hands, work over open vessels Do not work over (vertical laminar flow and open bench) or behind and over (horizontal laminar flow hood) an open bottle or dish.
Operator hair, hands, breath, clothing
Dust from skin, hair, or clothing dropped or blown into the culture	Wash hands thoroughly, wear a lint-free lab coat. Use a lab coat reserved exclusively for culture room. Tie back long hair, or wear a cap. Face away from work when talking is necessary.
Aerosols from talking, coughing, sneezing, etc.	Avoid working with a cold, or wear a mask.
Materials and Reagents
Solutions
Nonsterile reagents and media	Filter or autoclave solutions before use.
Inadequate sterilization procedures	Monitor performance of the autoclave with a recording thermometer or sterility indicator. Test or culture sterilized solutions if contamination is suspected.
Poor quality control at commercial supplier	Obtain media, buffers, sera only from reputable suppliers that certify quality and source.
Glassware and screw caps
Dust and spores from storage	Shroud caps with foil. Wipe bottles with 70% alcohol before taking them into the hood.
Instruments, pipettes
Unreliable sterility	Use individually wrapped, sterile, disposable supplies from a reputable supplier. Sterilize reusable items by dry heat before using them.
Contact with a nonsterile surface or some other material	Do not grasp any part of an instrument or pipette that will pass into a culture vessel.
Culture flasks and media bottles in use
Dust and spores from incubator or refrigerator	Use screw caps instead of stoppers. Swab bottles before placing in hood. Use secondary containment for plates.
Dirty storage or incubation conditions	Cover caps and necks of bottles with aluminum foil during storage or incubation. Clean out stores and incubators regularly.
Media under the cap and spreading to the outside of the bottle	Discard all bottles that show spillage on the outside of the neck. Do not pour.
Equipment and Facilities
Room air
Drafts, eddies, turbulence, dust, aerosols	Dedicate an appropriate space for tissue/cell culture Reduce traffic and extraneous activity. Wipe the floor and work surfaces regularly.
Laminar-Flow Biosafety Cabinets
Perforated filter	Check filters regularly for holes and leaks.
Dirty filter	Check the pressure drop across the filter.
Spillages, particularly in crevices or below a work surface	Disinfect around and below the work surface regularly. Let alcohol run into crevices.
CO2 humidified incubators
Growth of molds and bacteria on walls and shelves in a humid atmosphere	Clean regularly according to manufacturer’s specifications with appropriate disinfection.
Spores, etc., carried on forced-air circulation	Enclose open dishes in plastic boxes with close-fitting lids (but do not seal the lids).
Baths and other equipment
Contamination in water baths used to warm solutions	Clean and disinfect water bath regularly; wipe down vessels thoroughly with 70% alcohol before transferring to biosafety cabinet for use when dry.
Dust on gas cylinders, pumps, etc.	Consider using non-water based (bead) warming bath. Wipe with 70% alcohol before entry into culture room.
Importation of Biological Materials
Incoming Cell Lines
Contamination suspected at the source or during transit	Handle these cell lines alone, preferably in quarantine. Check for contamination by growing a culture for two weeks without antibiotics. Inspect for contamination visually, by phase-contrast microscopy and Hoechst/DAPI stain for mycoplasma.

Mycoplasma

Mycoplasma are a genus of bacteria that do not possess cell walls, and are therefore unaffected by antibiotics that limit bacterial growth by inhibiting cell wall formation. Their flexible morphology and cell size in the 0.15 to 0.3 µm range mean that they may escape standard cell culture filtration approaches that often use filters with 0.22 µm pores. Unlike most other bacterial contaminants, mycoplasma will not be apparent by casual inspection, and are difficult to detect by light microscopy due to their morphology and notably small size. Mycoplasma titer in culture can attain 10⁸ organisms/mL without causing turbidity of the medium. They do not usually kill the mammalian cells they infect, but significantly impact cultures by altering cellular metabolism, causing chromosomal aberrations, slowing cell growth, and interfering with cell attachment. In short, they are likely to dramatically influence the results of most experiments performed using affected cell lines.

Figure 2. Common DNA detection agents such as DAPI or Hoechst reveal the presence of mycoplasma in contaminated cultures (right) using fluorescent microscopy.

The varied sources of mycoplasma contamination in the laboratory can present a challenge. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique, in addition to introduction via contaminated supplements such as fetal bovine serum.  Mycoplasma are highly transmissible among neighboring contaminated cell cultures. Filtering media and buffers with membranes having pores 0.1 µm or less is necessary for mycoplasma treatment, as standard media filtration devices with pores of 0.22 or 0.45 µm will fail to exclude these small organisms.

Preventing, Detecting, and Eliminating Mycoplasma Contamination

Once mycoplasma contaminates a culture, it can quickly spread to other areas of the lab. Strict adherence to good laboratory practices is key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination. The three most popular methods for detection include mycoplasma culture, DNA staining method and PCR based detection. We offer a variety of solutions for  Mycoplasma Detection and Elimination. It is critical to establish a routine for mycoplasma screening of cultures even if you do not suspect the presence of this contaminant in the lab.

Preventing microbial contamination:  are antibiotics the answer? One in tissue culture is the routine use of antibiotics, either alone or in cocktails with antifungals. Just as with therapeutic antibiotics prescribed by physicians, the continuous or inappropriate use of antibiotics can lead to the development of resistant strains that are difficult to eradicate and may require the use of later-generation antibiotics that may be toxic to the cell cultures. Recent studies have raised additional concerns about the potential for altered gene expression in cultured cells in the presence of antibiotics.

Viral Contamination

Viruses are among the most difficult cell culture contaminants to detect in culture, requiring microscopy methods that would be impractical for most research laboratories. They can originate from the patient or host animal cell source, and several cell lines of biotechnological significance have been shown to contain endogenous retroviruses.  More often, cells become infected by viruses present in animal-derived materials used to culture them. The small size of viruses makes them very difficult to remove from media, sera, and other solutions of biological origin. However, because most viruses are host- and even tissue-restricted, this may limit their capacity for cross-species or cross-tissue infection. Although viruses may be more common in cell cultures than many researchers realize, they may or may not present a significant confounder to cell culture if they do not induce cytopathic or other adverse effects on cells.

Beyond their potential to impact cultured cells, a major concern of using virally infected cell cultures is the potential health hazard they pose for laboratory personnel. Special safety precautions should always be used when working with tissues or cells from humans or other primates to avoid possible transmission of viral infection (HIV, hepatitis B, Epstein-Barr, simian herpes B virus, among others) from the cell cultures to laboratory personnel. Institutional environmental safety officers should be consulted regarding procedures for working with potentially hazardous tissues, cultures, or viruses. For more on risk assessment for laboratory personnel working with cell culture, click here

Best practices for reducing the incidence of viral contamination of cell cultures:
*Limit the number of biological sources (suppliers, animals) from which cells are extracted.
*Select animals/cells which are less virus-susceptible.
*Source cells from repositories that perform virus testing and provide certification of virus-free cell lines.

Chemical Contamination

Any nonliving contaminant of cell culture is often classified as a ‘chemical’ contaminant.  These contaminants may originate from reagents or water used in media or buffers, or from equipment and supplies.  Examples include free radicals, metal ions, disinfectant/detergent residues, or even endotoxins that persist after upstream bacterial contaminants are no longer present.

Tips for preventing chemical contamination:
*Always use laboratory-grade water for preparing buffers and solutions, and resuspending lyophilized reagents.
*Any reusable labware must be rinsed exhaustively and air-dried—autoclaving will have no effect on detergent residue.
*Obtain media, supplements, and serum (FBS, FCS) exclusively from suppliers that provide endotoxin testing certification.

General Tips and Techniques for Preventing and Eliminating Contamination

Working Within the Biological Safety Cabinet

When working within the biosafety cabinet, it’s helpful to remember that the air-flow is key to helping maintain a sterile environment. Both the rear and front vents should be kept clear at all times to achieve effective airflow. Before any practical procedure, the cabinet should be stocked with all required materials in order to minimize the potential for transfer of contaminants on the operator’s sleeves and hands.

At least twenty minutes should elapse after airflow is initiated before placing items to be used in the cabinet. All items that enter the cabinet must be sprayed first with 70% (v/v) sterile alcohol and wiped with lint-free wipes to prevent dust and particulates from entering the cabinet. Ensure airflow is well-established before any tops or containers are opened, to allow the airflow to purge the work area of particulates that may have been introduced.

Figure 3. Working in Safety Cabinet

Pipetting and Prevention of Aerosols

Disposable plastic pipettes—also called serological pipettes, available in capacities from 1-100 mL—are indispensable tools for cell culture. These must be individually-wrapped to ensure sterility. Adherence to the following guidance can minimize contamination and safety risks associated with pipetting.

• Use automatic pipette aids, with each pipette aid restricted for use in a single cabinet.  To avoid contamination, disassemble and disinfect the pipette aid parts regularly. Ensure that pipette aid filters are kept well stocked and changed regularly.
• Use plugged pipettes (plugged at the top with cotton) whenever possible, especially when transferring medium.  Avoid drawing liquid into the pipette plug. If plug is accidentally wetted, change pipette aid filter immediately.
• Avoid touching serological pipette altogether by unwrapping partially, fitting end to pipette aid, then removing paper sleeve.
• To avoid generating contaminating aerosols, do not create bubbles in the medium or pipette.
  

Disinfection

Methods designed for the disinfection/decontamination of culture waste, work surfaces and equipment must not only minimize contamination risk, but be safe for lab personnel. Always wear appropriate personal protective equipment (PPE) such as gloves and eye protection when using concentrated forms of disinfectants. Selected gloves should protect against the substance being handled. Manufacturers’ charts will help to identify the best gloves for a given task. Major disinfectants fall into groups, and their relative merits can be summarized as follows:

Sodium Hypochlorite or Bleach

• Good general purpose disinfectant
• Active against viruses
• Corrosive against metals—should not be used on metal surfaces such as centrifuges
• Readily inactivated by organic matter and should therefore be made fresh frequently
• Use commercial bleach to create a 10% (v/v) solution in waste liquid or for surface disinfection

Alcohol (e.g. Ethanol, Isopropanol)

• Effective concentrations: 70% for ethanol, 60-70% for isopropanol
• Effective against bacteria. Ethanol is effective against most viruses, but not non-enveloped viruses
• Isopropanol is not effective against viruses
• Aldehydes are irritants, and their use should be limited

Phenolic-based disinfectants should be avoided, as they are not supported as part of the EU Biocidal Products Directive review program.

How to Perform Routine Monitoring of Healthy Cells

Monitoring cell cultures is an important part of everyday cell culture. This tutorial will help explain the basics of monitoring your cells, including what a healthy culture looks like, cell confluency, and the different phases of cell culture growth. This video will also show common indicators of bacterial and mycoplasma contamination.

Related Products

Culture Media	Fetal Bovine Serum	Mycoplasma Detection	Pipette Tips	Culture Flasks
Trypsin	Antibiotics	Hoechst Staining Dye	Ethyl Alcohol	Pipettors
Sterile Filtration	Authenticated Cell Lines	Isopropyl Alcohol

References

1. 2009. Identity crisis. Nature. 457(7232):935-936. https://doi.org/10.1038/457935b

2. Neimark J. 2015. Line of attack. Science. 347(6225):938-940. https://doi.org/10.1126/science.347.6225.938

3. Weiss RA. 1978. Why cell biologists should be aware of genetically transmitted viruses. Natl Cancer Inst Monogr. 48183-9.

4. Fogh J, Holmgren NB, Ludovici PP. 1971. A review of cell culture contaminations. In Vitro. 7(1):26-41. https://doi.org/10.1007/bf02619002

5. Merten O. 2002. 39(2):91-116. https://doi.org/10.1023/a:1022969101804

6. Freshney RI. Culture of Animal Cells. https://doi.org/10.1002/9780471747598

7. Fundamental Techniques in Cell Culture, Laboratory Handbook. [Internet]. Public Health England: Available from: https://www.phe-culturecollections.org.uk/media/101902/ecacc_lab_handbook.pdf

8. Emmett Barkley W. 1979. [4] Safety considerations in the cell culture laboratory.36-43. https://doi.org/10.1016/s0076-6879(79)58125-7

9. Ryu AH, Eckalbar WL, Kreimer A, Yosef N, Ahituv N. 2017. Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation. Sci Rep. 7(1): https://doi.org/10.1038/s41598-017-07757-w